Lyme disease, due to infection with the Ixodes tick-borne spirochete Borrelia burgdorferi (Bb) is the most common vector-borne disease in North America and a significant public health concern. Although usually amenable to antibiotic therapy, Bb infection can be followed by prolonged clinical symptoms, especially if treatment is delayed. Recent studies in mice reveal that under some circumstances, spirochetes can persist in connective tissue for extended periods after antibiotic treatment. Central to resolving the clinical issue of spirochete persistence after antibiotic therapy is an understanding of how Bb evades the normal host defenses that should complement antibiotic effects. Our preliminary findings of Bb interactions with phagocytes in vitro suggest that Bb outer surface membrane proteins (Osps) are highly mobile and that this feature, along with spirochete motility, facilitate its escape from near total engulfment by phagocytes, even after opsonization. We further show the feasibility of using multiphoton imaging to obtain time-resolved images of tick feeding and Bb movements within the murine host. This application will use in vitro imaging techniques and in vivo real-time multiphoton microscopy to further evaluate the contribution of Bb Osp mobility and spirochete motility to phagocyte evasion and Bb persistence within the mammalian host. Using a panel of mutant/transformant Bb with differential survival in mice, we will evaluate through confocal, electron and videomicroscopy the membrane mobility of their dominantly expressed Osp and the capacity of the Bb variants to escape engulfment by human monocytes and murine macrophages. We will then use real-time multiphoton imaging of live anesthesized mice to examine the requirement for OspC in tick-transmitted Bb infection and dissemination from the skin inoculation site, and effects of OspC deficiency on spirochete motility in vivo. Finally, we will use multiphoton microscopy to examine the changes in Bb motility patterns over time as it adapts to persist within mice, and the effects of antibiotics on Bb when administered during acute and chronic phases of infection. These studies will provide insight into the role of membrane protein mobility and spirochete motility in Bb evasion of immune destruction in vivo, and insight into how Bb may persist after antibiotic therapy for Lyme disease. Moreover, our results will also provide a powerful, well-characterized system for future real-time studies of the effects of specific genetic manipulations of the tick vector, the spirochete, and/or the mammal on tick-borne Bb infection.